RESEARCH ARTICLE Ribosomal L22-like1 (RPL22L1) Promotes Ovarian Cancer Metastasis by Inducing Epithelial-to-Mesenchymal Transition Nan Wu1, Jia Wei1, Yuhui Wang1, Jinyan Yan1, Ying Qin1, Dandan Tong2, Bo Pang1, Donglin Sun1, Haiming Sun1, Yang Yu1, Wenjing Sun1, Xiangning Meng1, Chunyu Zhang1, Jing Bai1, Feng Chen1, Jingshu Geng3, Ki-Young Lee4, Songbin Fu1*, Yan Jin1* 1 Laboratory of Medical Genetics, Harbin Medical University, Harbin, China, 2 Department of Pathology, Harbin Medical University, Harbin, China, 3 Department of Pathology, Third Affiliated Clinical Hospital, Harbin Medical University, Harbin, China, 4 Department of Cell Biology & Anatomy, University of Calgary, Alberta, Canada * [email protected] (YJ); [email protected], [email protected] (SF) Abstract OPEN ACCESS Citation: Wu N, Wei J, Wang Y, Yan J, Qin Y, Tong Double minute chromosomes (DMs) have important implications for cancer progression D, et al. (2015) Ribosomal L22-like1 (RPL22L1) because oncogenes frequently amplified on them. We previously detected a functionally Promotes Ovarian Cancer Metastasis by Inducing undefined gene amplified on DMs, Ribosomal L22-like1 (RPL22L1). The relationship Epithelial-to-Mesenchymal Transition. PLoS ONE 10 between RPL22L1 and cancer progression is unknown. Here, RPL22L1 was characterized (11): e0143659. doi:10.1371/journal.pone.0143659 for its role in ovarian cancer (OC) metastasis and its underlying mechanism was examined. Editor: Xin-Yuan Guan, The University of Hong DNA copy number and mRNA expression of RPL22L1 in OC cells was analyzed using data Kong, CHINA obtained from The Cancer Genome Atlas and the Gene Expression Omnibus database. An Received: August 5, 2015 immunohistochemical analysis of clinical OC specimens was performed and the relation- Accepted: November 6, 2015 ships between expression level and clinicopathological factors were evaluated. Addition- Published: November 30, 2015 ally, in vivo and in vitro assays were performed to understand the role of RPL22L1 in OC. Copyright: © 2015 Wu et al. This is an open access RPL22L1 expression was higher in OC specimens than in normal tissues, and its expres- article distributed under the terms of the Creative sion level was highly positively correlated with invasion and lymph node metastasis (P < Commons Attribution License, which permits 0.05). RPL22L1 over-expression significantly enhanced intraperitoneal xenograft tumor unrestricted use, distribution, and reproduction in any development in nude mice and promoted invasion and migration in vitro. Additionally, medium, provided the original author and source are RPL22L1 credited. knockdown remarkably inhibited UACC-1598 cells invasion and migration. Fur- ther, RPL22L1 over-expression up-regulated the mesenchymal markers vimentin, fibronec- Data Availability Statement: DNA copy number α α β analysis data can be obtained from oncomine.org tin, and -SMA, reduced expression of the epithelial markers E-cadherin, -catenin, and - (https://www.oncomine.org/resource/main.html) and catenin. RPL22L1 inhibition reduced expression of vimentin and N-cadherin. These results all the detailed steps are within the paper. All other suggest that RPL22L1 induces epithelial-to-mesenchymal transition (EMT). Our data relevant data are within the paper and its Supporting showed that the DMs amplified gene RPL22L1 is critical in maintaining the aggressive phe- Information files. notype of OC and in triggering cell metastasis by inducing EMT. It could be employed as a Funding: This study was supported by the novel prognostic marker and/or effective therapeutic target for OC. Programme for Changjiang Scholars and Innovative Research Team in University (Grant No. IRT1230 to YJ), the National Natural Science Foundation of China (Grant No. 81371617 to YJ), the Outstanding Youth foundation of Heilongjiang Province of China (Grant No. JC201215 to YJ). PLOS ONE | DOI:10.1371/journal.pone.0143659 November 30, 2015 1/14 RPL22L1 Promotes Ovarian Cancer Metastasis Competing Interests: The authors have declared Introduction that no competing interests exist. Ovarian cancer (OC) is the second most common gynecological malignancy and the first cause of death [1]. Cancer metastasis, rather than primary tumors, are responsible for most cancer deaths [2]. Owing to a lack of definitive early symptoms and appropriate markers for OC diag- nosis at an early stage, the majority of patients are diagnosed with late-stage OC accompanied with metastasis, which typically has a 5-year survival rate of < 30% [3]. It is critical to under- stand the molecular mechanisms involved in OC metastasis, and to determine efficient, spe- cific, and sensitive molecular targets that can be applied to metastasis diagnosis, prognosis, and individual treatment. Double minute chromosomes (DMs) are cytogenetic hallmarks of gene amplification [4]. DMs appear in various kinds of human cancer cells, but not in normal cells [5]. As extra-chro- mosomal elements carrying amplifications of genomic DNA sequences, DMs contribute to cancer formation and progression, oncogenes are frequently present in the amplified sequences and the proteins they encode are often over-expressed [6]. Examples of genes amplified on DMs include MYC in colon cancer [7], MYCN in neuroblastoma [8], EGFR in gliomas [9], and EIF5A2 [10, 11] in ovarian cancer [12]. As DMs are vehicles of amplified genes including many oncogenes, functional studies of genes that are amplified on DMs is a good way to explore can- didate oncogenes. Our team previously identified 3q26.2 as an origin of DMs in the human ovarian cancer cell line UACC-1598, and a series of genes were co-amplified on the same ovarian DMs, including MYCN, EIF5A2, and RPL22L1 [13]. Both MYCN and EIF5A2 play important roles in cancer progression. However, the relationship between RPL22L1 and cancer is not known. In this study, we showed that RPL22L1 is commonly over-expressed in clinical OC individu- als and its expression level is strongly related to tumor invasion and metastasis. An in vivo experiment showed that forced expression of RPL22L1 promotes intraperitoneal xenograft tumor development in nude mice, and enhances cell migration and invasion in vitro. Further- more, knocking it down with small interfering RNA (siRNA) inhibits migration and invasion in vitro. During this process, RPL22L1 over-expression resulted in elevated expression of mes- enchymal markers such as vimentin and α-SMA, and decreased expression of epithelial mark- ers, such as E-cadherin, α-catenin, and β-catenin, indicating that the induction of epithelial-to- mesenchymal transition (EMT) may explain the observed increases in motility and invasion ability for metastasis. Our data showed that RPL22L1 plays an important role in the process of OC metastasis. Materials and Methods Ethic statement This study were approved by the Ethics Committee of Harbin Medical University with the fol- lowing reference number, HMUIRB20150023. The ovarian cancer tissue microarrays (TMAs) for immunohistochemistry were purchased from US BIOMAX (ov951, ov1912, ov6161; Rock- ville, MD, USA) and Xin Chao (HOva-Can90PT-01; Shanghai, China). Both companies pro- vided ethical statements to confirm that the local ethics committees approved their consent procedures, all participants provided their written informed consents and all efforts had been made to protect patient privacy and anonymity. The ethical statements provided by companies and the protocol of experiment had been checked carefully and approved by Ethics Committee of Harbin Medical University (HMUIRB20150023). Four-week-old female BALB/c mice (spe- cific-pathogen-free) were purchased from SLAC (Shanghai, China) and housed in the Harbin Medical University Animal Laboratory. Mice were housed under standardized light-controlled PLOS ONE | DOI:10.1371/journal.pone.0143659 November 30, 2015 2/14 RPL22L1 Promotes Ovarian Cancer Metastasis conditions at room temperature (24°C) and 50% humidity, with free access to food and water. Animal experiments were performed in strict accordance with the recommendations in the Guidelines of Laboratory Animal Usage of Harbin Medical University. The protocol was approved by the Ethics Committee of Harbin Medical University (HMUIRB20150023) and all efforts were made to minimize suffering. Cell lines and cell culture Human ovarian cancer cell line UACC-1598, SKOV3, HO-8910, and HO-8910PM were pur- chased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). All cells were cultured following the methods described by the ATCC (Manassas, VA, USA), and were authenticated in 2012 at the Micro-read Genetics Company (Beijing, China) using a short tandem repeat analysis. Preparation of metaphase spreads and fluorescence in situ hybridization (FISH) analysis Cells were harvested for metaphase spread preparation according to the methods described in previous studies [14, 15] and were stained with Giemsa. Two BAC clones, GFP-RP11-355H10, which specifically covers the MYCN (amplified in UACC-1598 and located on the DMs [13]), and Cy3-RP11-726H11 for RPL22L1, were selected as DNA probes and hybridized to meta- phase spreads of cells as described previously [16]. Chromosomes were counterstained with DAPI (4, 6-diamidino-2-phenylindole). Images were captured using a Leica DM5000 B fluo- rescence microscope (Wetzlar, Germany), and analyzed using the MetaMorph Imaging System (Universal Imaging Corporation, West Chester, NY, USA). DNA copy number and gene expression analysis The Oncomine DNA